A study on the proliferation of Myzus persicae (sulzer) during the winter season for year-round production within a smart farm facility

In this study, we examined the feasibility of Myzus persicae proliferation through interrelationships with host plants in a smart farm facility during winter. We investigated aphid proliferation under an LED artificial light source and attempted to interpret aphid proliferation in relation to the net photosynthetic rate of the host plant, Eutrema japonicum. We observed that aphids continuously proliferated in the smart farm facility in winter without dormancy. The average number of aphids was greater under the 1:1 red:blue light irradiation time ratio, where the photosynthetic rate of the host plant was lower than under the 5:1 and 10:1 red:blue light irradiation time ratios. These results show that it is important to maintain a low net photosynthetic rate of the host plant, E. japonicum, in order to effectively proliferate aphids under artificial light such as in the case of smart farm facilities.

in a smart farm with controlled environment, and analyzed the cause of changes in aphid proliferation in relation to the host plant's net photosynthetic rate.

Plant cultivation and smart farm environment treatment
The host plant supplied as a food source for the aphids is E. japonicum, a perennial semishaded plant that has been observed to have aphids attached to and is suitable for cultivation in smart farm facilities with lower light than outdoor environments [32][33][34]. E. japonicum seedlings were cultivated from November 2021 to March 2022 in a smart farm by selecting similar sized plants with 3-5 aphids attached, and then transplanting one individual per pot. In the three chambers installed in the smart farm (Parus Co., Shanghai, China), pots with transplanted E. japonicum (12 pots per chamber) were placed, and the day length was set to 16 h. The smart farm we used has been owned the ecology lab in Kongju National University and was permitted for use in experiments.
The size of the smart farm was 360 cm (W) × 60 cm (L) × 230 cm (H). The size of each chamber positioned in the smart farm was 120 cm (W) × 52 cm (L) × 41.5 cm (H), and their walls were painted white to increase the light reflectance (Fig 1). In order to observe the aphid proliferation response according to light quality, different ratios of red (R, 660 nm) and blue (B, 450 nm) light efficient for plant photosynthesis was used for examining light quality inside the smart farm by controlling irradiation time of them [22,[35][36][37][38][39][40]. The irradiation time rates of R and B lights in the LED panels were set to 1:1, 5:1, and 10:1 (Table 1). In this case, the

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ratios of the amount of blue light / red light was 0.64 in RB(1:1), 0.13 in RB(5:1), 0.06 in RB (10:1), if calculated, because the number of LED units of red light was 33, and the number of LED units of blue light was 21 per chamber.
For E. japonicum cultivation, sand with the same particle size was placed in a circular pot of 15 cm (H) × 12 cm (D). The sand was mixed with bed soil (Heungnong bio, Monsanto Korea, Seoul, South Korea) at a ratio of 99.5:0.5 to supply the pot with nutrients. Water was supplied at intervals of 2-3 days so that the soil would not dry out, and the temperature and relative humidity were controlled using a thermo-hygrostat data logger LCSEMS (Parus Co., Shanghai, China). During the experimental period, light intensity in the smart farm was 101.44 ± 8.07 μmolm -2 s -1 , temperature was 11.15 ± 4.24˚C, relative humidity was 50.84 ± 8.26%, and CO 2 concentration was 355.18 ± 12.24 ppm (average ± standard deviation for all values).

Measurement of aphid population
We cultivated E. japonicum with M. persicae placed in each chamber for one month to allow for acclimatization to the internal environment of the smart farm and then observed the number of aphids per leaf in each individual plant every month from December 2021 to March 2022. During this period, to determine whether there is a difference in the number of aphids depending on the location on the leaf, the number of aphids on the upper side (UPP) and lower side (UND) of the leaves was observed. Adult individuals of Myzus persicae were counted and divided into two groups, marking different external morphology: alate aphids (W) with a pair of wings, and apterous aphids (S) without wings.

Measurement of the ecophysiological response of the host plant
To determine the change in the number of aphids in relation to the net photosynthetic rate (Pn; expressed in μmol m -2 s -1 ) of the host plant, we measured the Pn of leaves to which aphids had been attached. Measurements were taken 39 times per chamber between 10 a.m. and 2 p. m. for a day in March 2022 using the photosynthesis measuring equipment LCi-SD Ultra Compact Photosynthesis System (ADC BioScientific Ltd., Hoddesdon, UK), which can measure photosynthetic factors, such as Pn.
To understand whether the leaf toughness of host plants affects the number of aphids on it, aphid-attached E. japonicum leaves with signs of feeding activity were sampled. Per chamber, nine leaf disks with a diameter of 5 mm were collected, and their dry weight was determined to calculate the leaf density, which is the leaf dry weight per constant area. It is known that increased leaf density increases leaf toughness [41], in turn affecting herbivorous pressure, e.g., by aphids, on the host plant.

Statistical analysis
Statistical tests to compare among the light conditions for the Pn of E. japonicum and the number of leaves with M. persicae were performed using the one-way ANOVA test (p�0.05), and the post hoc test was performed using the Fisher LSD method [42]. Factor analysis was performed to determine the relationship among the observed number of aphids on the upper side (UPP) and lower side (UND) of leaves, number of alate aphids (W) and apterous aphids (S), and net photosynthetic rate (Pn) of E. japonicum leaves attached with aphids [42]. When analyzing the factors, standardized data were used to eliminate errors caused by different units for each variable [42]. Statistical analyses were performed using Statistica (version 7) statistical package (StatSoft Inc., Tulsa, USA).  Table). However, there was no difference in leaf density in relation to light quality (Fig 4).

Results
Factor analysis using the number of aphids and the Pn of E. japonicum measured in March showed that the number of aphids was affected by light quality and Pn ( Fig 5 and Table 2). When the distributed factor scores of RB(1:1), RB(5:1), and RB(10:1) were divided into three

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groups, their distribution positions were separated into factors 1 and 2 ( Fig 5). Considering that a variable with factor loading of 0.3 or more is a major variable for factor 1 or 2 [43], the major variables affecting the distribution of the factor scores were UPP, UND, W, S, and Pn ( Fig 5 and Table 2). Therefore, the characteristics of aphids were affected by the Pn of E. japonicum.

Discussion
M. persicae was continuously observed from December to March, which is the winter season in temperate Asia, under the light conditions of RB(1:1), RB(5:1), and RB(10:1) in the smart farm (Fig 2A). On average, the number of aphids per month increased by approximately 32-fold compared to the number of early aphids. However, in the wild environment within temperate Asia of northern hemisphere, aphids generally appear from late March or early April and have been observed to be active until late October or early November [44][45][46]. This result was inconsistent with the previously reported life cycle of M. persicae in the wild, as this species lays eggs in November and winters them until March-April of the following year [44]. Therefore, when M. persicae were supplied with E. japonicum as a host plant in a smart farm, we observed continuous proliferation over time irrespective the aphid life cycle in the wild environment.
In this regard, M. persicae may accomplish continuous asexual reproduction under a stable climate and food supply [47]. Acyrthosiphon pisum, which is closely related to M. persicae, is known to induce asexual reproduction by thelytokous parthenogenesis at high temperatures [48,49]. In general, because the numbers of prey and predators in complex environments exhibit continuous oscillation [50], it is possible that M. persicae continuously proliferated in the smart farm due to the blocking of access of their predators, the suitable artificial light conditions, and the maintenance of stable temperature inside the facility for their proliferation (average of 11˚C).

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The number of aphids under RB(1:1) was higher in December and March than in January and February (Fig 2B). In contrast, it was higher under RB(5:1) in January and February than in December and March (Fig 2B). Under RB(10:1), it was highest in February ( Fig 2B). Changes in the number of aphids over time are generally associated with the temperature, aphid growth period, aphid sexual/asexual reproductive period, aphid life span, and alate forms of plant-to-plant migration [44,47,48,51]. The different oscillating patterns of the number of aphids according to these light conditions imply that the number of aphids may also be affected by the light conditions.
The number of aphids and the Pn of the host plant E. japonicum showed opposite results depending on the light conditions (Figs 2 and 3). The number of aphids was highest under RB (1:1) and lowest under RB(10:1) (Fig 2B). Conversely, the Pn of E. japonicum was lowest under RB(1:1) and highest under RB(10:1) (Fig 3A). Therefore, with an increasing RB ratio, the Pn increased, whereas the number of aphids decreased. Additionally, although there were no differences in leaf densities between light conditions, the number of aphids attached per E. japonicum plant was the highest under RB(1:1) and lowest under RB(10:1), indicating that aphids prefer host plants with low Pn, regardless of the leaf toughness (Figs 3B and 4). This result may be attributed to the photosynthetic capacity of E. japonicum according to light quality and the complex responses to the predator-prey relationship between aphids and E. japonicum.

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The Pn of plants can be affected by the ratio of red to blue light [38][39][40], and plants respond to herbivory through photosynthetic products [52]. Plant Pn is influenced by various physiological variables. Therefore, it is possible to manage environmental changes by actively controlling photosynthesis using various physiological variables within the limited photosynthetic capacity that can potentially occur when the environment changes [53]. However, because

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photosynthetic capacities can be altered by light stress [54], in environments where photosynthetic capacities are low, plants may find it difficult to cope with such situations by controlling their Pn when environmental pressures such as herbivory occur.
The light quality also directly can affect producing their secondary metabolites as well as Pn [55]. The blue light enhanced total phenolic contents of Stevia rebaudiana [56], and in Brassica juncea, the glucosinolate contents (GSLs) were directly affected on the white, red and blue single light [57]. These reports provide the insight the secondary metabolites, mainly using plant defense on their herbivores, of E. japonicum were also directly affected on the light quality.
The host plants belonging to the Brassicaceae family, which includes E. japonicum, respond to herbivory through primary and secondary metabolites [58]. Starch is one of the primary metabolites [52]. Arabidopsis thaliana, another member of the Brassicaceae family, transports sugar to tissues damaged by M. persicae aphids, resulting in a reduction in the number of aphids by interfering with the herbivory of M. persicae through osmotic effects [52].
Brassicaceae plants also produce secondary metabolites, such GSLs, for chemical protection [59][60][61]. A. thaliana accumulated GSLs in their leaves that were damaged by M. persicae, and when GSL production was high, the number of aphids decreased [62]. Therefore, the higher the Pn of E. japonicum when infected with aphids, the more primary and secondary metabolites may be produced, resulting in a more efficient defense system against aphids. In the present study, under the light conditions of RB(5:1) and RB(10:1) (compared to RB(1:1)), E. japonicum could more easily attain photosynthetic capacity and subsequently improve the Pn more easily to defend against herbivory by aphids. Therefore, there were fewer aphids sucking the sap of E. japonicum and fewer leaves attached to the aphids (Fig 3B).
Factor analysis revealed that the Pn of E. japonicum had a negative effect on the number of apterous aphids and the number of aphids on the upper and lower sides of E. japonicum leaves ( Table 2); considering that the distribution ranges of the factor scores for each light condition were divided, there was a difference in these effects depending on the light quality (Fig 5).
Given that the proportion of alate aphids to apterous aphids observed during the study period was very small (5.34%) and the number of alate aphids was not related to Pn, regardless of the settled position of aphids on the E. japonicum leaf, Pn is considered to have a negative effect on apterous aphids rather than alate aphids. In this reason, the apterous form is thought to the main aphid form that causes herbivorous damage to E. japonicum rather than the alate form.
When the number of aphids attached to the lower side of the leaf increased, the number of alate aphids also increased (Table 2). However, because Pn was not related to the number of alate aphids, this result may be caused by the increase in aphid population density due to the production of apterous aphids on the lower side of the leaf [51].

Conclusions
In summary, unlike general crop production facilities, smart farms are sealed from the external environment and are not affected by the weather, and the internal environment can be

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artificially controlled. Maintaining a constant temperature inside a smart farm during winter can sustain the growth of the perennial E. japonicum. Therefore, using E. japonicum as a host for aphids, even in winter, allows M. persicae to continuously produce progeny rather than wintering with eggs, as in the wild environment, thereby enabling the growth of aphids. The number of aphids increased with lower Pn, regardless of the leaf density of the host plant E. japonicum. The Pn of E. japonicum was negatively affected by the number of apterous M. persicae aphids and the number of aphids on the upper and lower side leaves of E. japonicum. Moreover, the number of alate aphids was positively affected by the number of apterous aphids attached to the lower side of the leaves rather than Pn.
Therefore, it is possible to mass-produce aphids for augmentative biocontrol agents in the winter season by maintaining a constant temperature within a smart farm with limited access to predators and supplying suitable light quality that can lower the Pn of the perennial host plant, E. japonicum.
Supporting information S1 Table. The